1. Field of the Invention
This invention relates to biopolymer salts having low levels of endotoxin and to biopolymer compositions thereof. The biopolymer salts of this invention are particularly useful as parenteral implants. The invention also relates to methods of purifying biopolymer salts, such as alginates and biogums, to prepare the novel biopolymer salts having low endotoxin content.
2. Background of the Invention
Materials which are to be used parenterally in the body must be essentially free of pyrogens, which are materials that induce fever by triggering an immune response. Introduction of pyrogenic materials into the body can produce a reaction severe enough to produce shock or even death. An important pyrogenic material is the lipopolysaccharide endotoxin which exist as a component of the cell walls of gram negative bacteria. These endotoxins are released in large quantities when the gram negative cells undergo lysis. Materials which come into contact with water having high gram negative bacterial counts can be expected to contain significant quantities of lipopolysaccharide endotoxin. Although this does not pose a problem for compositions that are administered orally, it is unacceptable for parenterally administered compositions.
Lipopolysaccharide endotoxin is not a living material and cannot be deactivated by common sterilization techniques such as autoclaving. While gamma irradiation and dry heat sterilization techniques do destroy endotoxin, these techniques also may destroy or damage many other compounds in the composition. Therefore, many sterile products can contain significant levels of endotoxin unless the endotoxin is specifically removed or deactivated.
In addition, because the lipopolysaccharide originates from gram negative bacteria, non sterile material that was originally endotoxin free can become contaminated with endotoxin as the organisms multiply. Endotoxin free products also can become contaminated after contacting surfaces containing endotoxin; these are primarily surfaces that have contacted water. Thus a composition which is to be administered parenterally must be free of endotoxin and must also be sterile to avoid regeneration of lipopolysaccharide endotoxin.
Biopolymer products such as alginic acid and its salts, gellan gum, and xanthan gum are known for use in a number of pharmaceutical applications, including for example, in sustained release pharmaceuticals that are orally ingested. However, these prior biopolymer products have had an endotoxin level that is not suitable for parenteral administration. For parenteral use, the endotoxin level of biopolymer salts should be less than about 100 endotoxin units per gram of biopolymer, and preferably less than 50 endotoxin units per gram of biopolymer salt. It would be highly desirable to provide biopolymer salts having an endotoxin content sufficiently low such that the biopolymer salts are suitable for parenteral administration.
The present invention is directed towards biopolymer salts that are suitable for parenteral use. In particular, this invention relates to biopolymer salts, such as alginates or biogums, having water-soluble polysaccharides that are biologically-produced and having an endotoxin content less than about 100 endotoxin units per gram. The invention is also directed to alginate or biogum compositions comprising the biopolymer salt of this invention and a pharmaceutically acceptable solvent.
Another embodiment of this invention relates to methods for preparing the biopolymer salts and compositions thereof that are suitable for parenteral use. In particular, one method comprises the steps of (i) contacting an aqueous solution of a biopolymer salt with a hydrophobic material to adsorb endotoxin on said material; and (ii) precipitating a biopolymer salt having an endotoxin content less than about 100 endotoxin units per gram from the solution by mixing a water miscible organic solvent with the solution. In yet another embodiment of the method of this invention, the step of precipitating may be replaced by the step of extracting endotoxin from the aqueous solution with a water immiscible organic solvent. These methods advantageously provide biopolymer salts and biopolymer compositions that have an endotoxin content of less than 100 endotoxin units per gram. These methods can be applied to a wide variety of biopolymer salts comprising water-soluble polysaccharides, including not only the alginates and biogums mentioned above, but also chitosan, chitan, carrageenan, agar, welan gum, S-657 gum, rhamsan gum, carboxymethylcellulose, and chemical substitutions of carboxymethylcellulose, among others. The novel biopolymer salts and compositions thereof are highly suitable for use as parenteral implants. They may also be used, for example, to supplement natural lubricating fluids, to coat catheters, to thicken parenteral injections, to provide tissue bulking, and for cell encapsulation techniques.
Endotoxin levels typically are measured using the Limulus Amoebocyte Lysate (LAL) test method. There are several variations of this test method in common use (e.g., Gel-Clot Endpoint, Chromogenic LAL, Kinetic-Chromogenic LAL) which produce a visual or color response in proportion to the amount of endotoxin present. Endotoxin levels are measured in endotoxin units (eu).
The biopolymer salts and biopolymer compositions of the present invention have endotoxin levels of less than about 100 eu per gram of biopolymer salt on a dry basis. Preferably, the biopolymer salts and biopolymer compositions of the present invention have endotoxin levels less than about 50 eu per gram, and more preferably, less than about 20 eu per gram.
The biopolymer salts of this invention are water-soluble polysaccharides that are either exuded by, or are extracted from, living organisms. Alginates are salts of alginic acid, which is a copolymer composed of D-mannuronic acid and L-guluronic acid units. These units typically exist as blocks of polymannuronic acid, blocks of polyguluronic acid or blocks of alternating mannuronic and guluronic acid units. The arrangement and relative amounts of mannuronic and guluronic acid are determined primarily by the source from which the alginate is manufactured. For example, most commercial alginate salts are produced by extraction from brown seaweeds. Alginate produced from Macrocystis pyrifera has a mannuronic to guluronic unit ratio (M/G ratio) of about 1.56:1 while alginate produced from Laminaria hyperborea has an M/G ratio of about 0.45. Monovalent salts (sodium or potassium salts) of alginate are typically water soluble while divalent salts (calcium, barium), polyvalent salts (iron, aluminum, etc.) and alginic acid form water insoluble gels or solids. Alginates are commercially available from ISP Alginates (San Diego, Calif.).
Biogums are salts of complex organic acids and are produced by fermentation of microrganisms. Gellan gum refers to the extracellular polysaccharide obtained from microorganisms of the species Sphingomonas elodea, in a suitable nutrient medium. Similarly, xanthan gum is a hydrophilic polysaccharide which is obtained by fermentation of microorganisms of the genus Xanthomonas, in a suitable nutrient medium. Gellan gum and xanthan gum are useful viscosifying agents. Gellan gum is also useful as a gelling agent. Depending on the biogum, monovalent salts (sodium or potassium salts) typically, but not necessarily, will render the biogum water soluble while divalent salts (magnesium, calcium, barium) and polyvalent salts (iron, aluminum, etc.) in the biogum may, but not necessarily, form water insoluble gels or solids.
The biopolymer employed in this invention is an alginate or biogum. The alginate is a salt of alginic acid, whereas the biogum is a salt of a complex organic acid, typically with a long polymer chain that increases viscosity.
Most preferably, with regards to alginates, the salt is sodium alginate. The alginate will typically have a ratio of mannuronic acid to guluronic acid of about 0.3:1 to about 2:1. In general, high mannuronic acid alginates have a ratio greater than 1 while high guluronic acid alginates have a ratio less than 1.
An example of a preferred source of alginate which maybe used in preparing the purified alginates of the present invention is xe2x80x9cKELTONExe2x80x9d, which is available from ISP Alginates (San Diego, Calif.). KELTONE LVCR is obtained from Macrocystis pyrifera giant kelp, and is a high mannuronic acid content alginate, having a ratio of mannuronic acid to guluronic acid of about 1.56:1.
Typically, commercially available KELTONE LVCR has an endotoxin level in the range from about 30,000 eu per gram to about 60,000 eu per gram. Pharmaceutical compositions for parenteral administration typically should have no more than about 100 eu per gram. Consequently, before KELTONE LVCR may be used in used in a parenteral application, the level of endotoxin must be reduced substantially. This invention provides a method for reducing the level of endotoxin in known salts of alginic acid, such as KELTONE LVCR, to below about 100 eu per gram.
Two examples of a preferred source of biogum which may be used in preparing the purified biogum of the present invention are xe2x80x9cGELRITExe2x80x9d gellan gum, derived from the microoganism Sphingomonas elodea, or xe2x80x9cKELTROL Txe2x80x9d xanthan gum, which is derived from the microorganism Xanthanomas campestris. Both biogums are available from Kelco Biopolymers (San Diego, Calif.). Typically, commercially available GELRITE gellan gums or KELTROL T xanthan gums are produced from gram negative bacteria and consequently are found to have endotoxin levels over 1,000,000 eu per gram. Removing these exceedingly high loads of endotoxin from the biopolymer in a commercially efficient manner can be particularly challenging. As described above in regards to alginates, pharmaceutical compositions for parenteral administration typically should have no more than 100 eu per gram. Consequently, before biogums may be used in a parenteral application, the level of endotoxin must be dramatically reduced. This invention provides a method for reducing the level of endotoxin in known biogums to below 100 eu per gram.
The lipopolysaccharide endotoxin molecular structure consists of a lipid head and a polysaccharide tail. Without being bound by theory, it is believed that the lipid portion of the polymer induces the pyrogenic response and that removing or disrupting the lipid portion may eliminate the induced response. Since the polysaccharide tail of the endotoxin is similar in molecular structure to the biopolymer, separation of the endotoxin from the biopolymer salt is not a simple matter.
A number of techniques, which are disclosed in the literature, are employed in the pharmaceutical industry to remove endotoxin from materials. However, many of these methods would also destroy or otherwise interact unfavorably with the biopolymer molecule, making such techniques inappropriate for depyrogenation of biopolymer compositions.
The present method of this invention uses the combination of two techniques to obtain the heretofore unavailable purified biopolymer salts. In particular, it has been discovered that when these techniques are used in combination, the level of endotoxin in alginates and biogums can be reduced to less than about 100 eu per gram.
This method for preparing a biopolymer composition comprising a salt of a biopolymer having an endotoxin content less than about 100 endotoxin units per gram comprises the steps of (i) contacting an aqueous solution of a biopolymer salt with a hydrophobic material to adsorb endotoxin on said material; and (ii) precipitating the biopolymer salt having an endotoxin content less than about 100 endotoxin units per gram from the solution by mixing a water miscible organic solvent with the solution.
Generally, the starting aqueous solution will have an alginate or biogum concentration of about 0.5 to about 5 percent by weight of the solution. Most preferably, the aqueous solution is a mixture of alginate and water, or biogum and water.
As noted above, one element of the method of this invention includes the adsorption of the endotoxin onto hydrophobic materials. Without wishing to be bound by theory, it is believed that the lipid end of the endotoxin molecule is attracted to the hydrophobic material. Biopolymer salts, which are polysaccharide polymers, lack this hydrophobic character. Therefore, they are not believed to adhere to the hydrophobic material.
Preferred hydrophobic materials for use in this invention include, for example, polystyrene, polypropylene, fluorocarbon polymers such as Dupont""s xe2x80x9cTEFLONxe2x80x9d and the like. Polypropylene and polystyrene are most preferred.
If the hydrophobic surface is used in the form of a filtration membrane, it may also be possible to physically filter cells or cell fragments from the solution, thereby further reducing the amount of endotoxin. Filtration membranes also advantageously provide an extremely large surface area for adsorption of endotoxin onto the hydrophobic surface.
If a hydrophobic filtration membrane is employed, then preferably the membrane will have a pore size of about 1.0 microns to about 0.1 microns. A particularly preferred hydrophobic filtration membrane is a polypropylene membrane having a pore size of about 0.2 microns.
Alternatively, the hydrophobic surface can be in the form of hydrophobic resins. Hydrophobic resins can provide efficient contact with high volumes of endotoxins. Hydrophobic resins provide additional advantages over filter membranes in that the hydrophobic resins can be regenerated and reused and can be easily increased in quantity to contact greater volumes of biopolymers. Hydrophobic resins are also more suitable for materials such as biogums, which are difficult to filter through the hydrophobic filter membranes due to viscosity and/or the length of the polymer chains.
As with the hydrophobic filter membranes, the hydrophobic resins can be varied in size to provide contact with a greater surface area. A particularly preferred hydrophobic resin bead of this invention is less than 0.5 mm in diameter and is comprised of polystyrene divinyl benzene.
If the hydrophobic resin method of purification is employed, a preliminary step may be required before contacting the biopolymer salt with the hydrophobic resin. The pH of the solution should first be raised to increase lipopolysaccharide endotoxin solubility before contact with the hydrophobic resin is made. Preferably, the pH is raised to at least 9 by addition of NaOH, KOH or other bases known to those skilled in the art. After contacting, the hydrophobic resin beads are sieved from the solution, and the pH is preferably adjusted back to neutral for the second purification technique of the method, as described below.
While the endotoxin lipopolysaccharide is known to bind to hydrophobic materials such as activated charcoal, polypropylene, and polystyrene, experiments using this contacting technique alone did not successfully produce a biopolymer salt having an endotoxin content of less than 100 eu per gram.
It has been discovered, however, that a highly purified biopolymer salt can be obtained by mixing the aqueous solution that was contacted with the hydrophobic surface with a water miscible organic solvent. This second step results in the precipitation of the highly purified biopolymer salt from the solution.
Without being bound to theory, it is believed that the lipid portion of the endotoxin molecule provides the molecule with solubility in hydrophobic liquids, such as hexane or methyl tert-butyl ether, or in partially hydrophobic liquids such as alcohols. In contrast, biopolymer salts will precipitate in water miscible hydrophobic liquids such alcohols and ketones which have suitably low dielectric constants. Thus, the endotoxin in the aqueous solution is separated from the biopolymer salt.
The water miscible organic precipitation solvent is selected from the group consisting of alcohols, ketones, aldehydes and mixtures thereof. Preferably, it is a low molecular weight alcohol. More preferably it is isopropyl alcohol, methanol, ethanol or acetone.
The water miscible organic solvent is typically mixed with the hydrophobic material treated aqueous solution at a volume ratio of about 1:1 to about 6:1. The mixture is held at a temperature and for a time sufficient to allow for the precipitation of the purified biopolymer salt.
After precipitation, the biopolymer salt may be dried to remove the solvent. A preferred technique of drying is low temperature oven drying (40-80xc2x0 C.). Other suitable drying techniques include, but are not limited to, lyophilization and spray drying. The biopolymer salt can also be reconstituted in an acceptable solvent. A particularly preferred solvent is water.
In another embodiment of this invention, the method may be carried out by replacing the precipitation step with a liquid-liquid extraction using a water immiscible solvent. An exemplary water immiscible solvent includes hexane or methyl tert-butyl ether.
The method of this invention may include additional steps as desired. For example, in regards to alginates, it is preferable to treat the starting aqueous solution with an oxidation agent, such as 100 ppm NaOCl, to destroy the polyphenols and thereby remove color from the alginate. Alternatively, activated carbon can be contacted with the aqueous solution in lieu of the NaOCl. Most preferably, the activated carbon is added after the NaOCl to adsorb polyphenols and remove any residual NaOCl, which can breakdown alginates in storage. In addition, the step of contacting with a hydrophobic material and/or precipitation may be conducted multiple times if desired. If a filtration membrane is used it may be preferable to pass the aqueous solution through membranes having different pore sizes. For example, it may be preferred to employ a 10 micron hydrophobic membrane followed by a 0.2 micron membrane. Using this sequence will tend to improve the capacity of the smaller pore sized filter. Similarly, if hydrophobic resins are employed, the size and quantity of the hydrophobic resin beads can be varied, or layered, to maximize the available surface area available and obtain the desired level of contact.
The method of this invention is particularly advantageous because the purified biopolymer salt may be prepared using a commercial scale manufacturing process.
The biopolymer salts of this invention may be used to prepare biopolymer compositions that are suitable for parenteral administration to a patient. This may be accomplished by dissolving a biopolymer salt of this invention having an endotoxin level less than or equal to about 100 eu per gram in a pharmaceutically acceptable solvent. Preferably, the pharmaceutically acceptable solvent is water for injection. Water for injection is a certified sterile, endotoxin free and particulate free pharmaceutical grade of deionized water.
The concentration of biopolymer salt in the composition may vary between about 0.5 weight percent and about 5 weight percent, based upon the total weight of the solution. Preferably, the concentration of biopolymer salt is between about 2 weight percent and about 4 weight percent.
Many of the biopolymer compositions of this invention also include gels prepared by adding a gelling agent to the above-described biopolymer composition. These gels are suitable for parenteral administration. The gels may be made into any desired shape. For example, the gels may be made in the form of beads, sheets or filaments which may be administered to a patient. Preferred gelling agents include divalent or trivalent cations. It also may be possible to incorporate a pharmaceutically active component into the biopolymer gel before administering it to a patient.
The amount of gelling agent that may be added to the biopolymer solution to form a suitable gel may vary depending upon the concentration of biopolymer in the solution as well as upon the particular gelling agent employed. Preferably, the gelling agent is added as an aqueous solution in which the gelling agent is present at a concentration range from about 0.5% to about 10%.
The examples which follow are intended to illustrate certain preferred embodiments of the invention, an no limitation of the invention is implied.